Heretofore, in identifying target small objects which are labeled with a fluorescent substance or the like, this is performed using a flow cytometer 160 such as shown in FIG. 18. The flow cytometer 160 has a passage 151 whereby a fluid containing the target small object flows along the interior. The passage 151 fluidicly couples a water flow tank 157 to a transport pump 158, which, in turn, is fluidicly coupled to a measuring passage 152 formed from a material with transmittance.
At a measuring position on the outside of the measuring passage 152 there is provided a light source 154 which shines excitation light of one type over a wide region on this measuring passage 152, and a light receiving section 153 which receives the fluorescence generated from fluorescent material which is provided on target small objects existing in the illumination region. The strength of the received light is analyzed by an analyzing section 155, to thereby detect the presence or absence of the target small objects. In the passage 151 there is provided in addition, the transport pump 158, diverter valves 159 and 161, and a waste tank 163. Reference symbol 156 denotes a suction mechanism, which is for drawing up the liquid containing the small objects to inside the passage 151 from a container 21 provided on the outside, via a nozzle 162.
Incidentally, conventionally there is a device for detecting the presence/absence of the target small objects which are labeled by the one type of labeling substance, and by using the flow cytometer, it is possible to measure the presence/absence thereof. However, recently, for the determination or analysis of base sequences of DNA, there is a growing requirement to label target substances of a large number of kinds.
In order to label target substances of a large number of kinds in this manner, it is not sufficient to merely use labeling substances of a plurality of different kinds for the small objects, and it is necessary to also specify the quantity ratio (molar ratio) in labeling (International Patent Publication WO 00/5357, Masayuki Machida et. al.). At this time, in order to obtain information from the labeling substances for each of the small objects, it is necessary to be able to independently measure the many small objects one at a time, so that information from the many kinds of labeled small objects is not measured overlapped.
Furthermore, in the case where several kinds of labeling substances are used, it is necessary to measure all of the various pieces of information obtained from the same small object.
However, if for the same small object, several kinds of excitation light are shone simultaneously all together, and the several kinds of light are received simultaneously all together without exception, since the plurality of light source sections and the plurality of light receiving sections are provided centralized, then in the measuring position, the construction of the optical measuring unit is centralized and complicated. Moreover, due to the influence of light shining from the various light source sections, there is the possibility that reception of the necessary light may be obstructed. Therefore, there is the problem that the number of kinds of labeling substance which one small object can have is naturally limited.
Moreover, with a device for small objects labeled by fluorescent substances of several kinds, which shines excitation light of several kinds onto a comparatively wide region at one measurement position of the passage, and receives the light of several kinds from the small objects which are in the comparatively wide region, there is a problem in that it is not possible to individually identify the small objects of several kinds which are labeled by the several kinds of labeling substances.
Therefore, the present invention addresses the aforementioned problems with a first object of providing a small object identifying device and its identifying method in which, by paying attention to the label elements which the respective small objects have rather than paying attention to each small object and measuring at once all of the label elements on the small object for each small object, and resolving for each of the label elements of a small number of kinds compared to the kinds of small objects, and providing a time difference and measuring, then the structure for the respective measurement points can be simplified as much as possible. Furthermore the amount of light to be shone can be reduced and the adverse affect on the received light can be reduced as much as possible, and the small objects can be identified, surely and with high reliability.
A second object is to provide a highly accurate small object identifying device and its identifying method which, by providing a time difference at two or more measurement points for a single small object and measuring, can measure the various different attitudes and directions between the measurement points for a single small object, and which can measure the label information from the respective small objects without exception.
A third object is to provide a highly accurate small object identifying device and its identifying method which, by dispersing a large quantity of small objects and providing a relative temporal relationship at two or more measurement points and then measuring, the influence of overlapping of the small objects can be eliminated as much as possible.
A fourth object is to provide a small object identifying device and its identifying method which can identify easily, reliably and efficiently a large number of small objects from several tens, to several hundreds, to several thousands, to more than several tens of thousands, without stopping in the case of identifying several kinds of small objects.
A fifth object is to provide a small object identifying device and its identifying method which is applicable to analysis, diagnosis and inspection of DNA, immune systems, proteins, amino acids, and biopolymers such as sugars.
A sixth object is to provide a small object identifying device and its identifying method with flexibility and diversity which can flexibly correspond according to the processing purpose.